Biomedical subjects

J B Burch

Publications and source records attributed to J B Burch.

At least 37 records · Page 2Linked to original sources

PubMed1995

DNA-binding specificity of the PAR basic leucine zipper protein VBP partially overlaps those of the C/EBP and CREB/ATF families and is influenced by domains that flank the core basic region.

The PAR subfamily of basic leucine zipper (bZIP) factors comprises three proteins (VBP/TEF, DBP, and HLF) that have conserved basic regions flanked by proline- and acidic-amino-acid-rich (PAR) domains and functionally compatible leucine zipper dimerization domains. We show that VBP preferentially binds to sequences that consist of abutted GTAAY half-sites (which we refer to as PAR sites) as well as to sequences that contain either a C/EBP half-site (GCAAT) or a CREB/ATF half-site (GTCAT) in place of one of the PAR half-sites. Since the sequences that we describe as PAR sites and PAR-CREB/ATF chimeric sites, respectively, were both previously described as high-affinity binding sites for the E4BP4 transcriptional repressor, we infer that these sequences may be targets for positive and negative regulation. Similarly, since the sequences that we describe as PAR-C/EBP and PAR-CREB/ATF chimeric sites are known to be high-affinity binding sites for C/EBP and CREB/ATF factors, respectively, we infer that these sites may each be targets for multiple subfamilies of bZIP factors. To gain insights regarding the molecular basis for the binding-site specificity of PAR factors, we also carried out an extensive mutational analysis of VBP. By substituting five amino acid residues that differ between the Drosophila giant bZIP factor and the vertebrate PAR bZIP factors, we show that the fork region, which bridges the basic and leucine zipper domains, contributes to half-site sequence specificity. In addition, we report that at least two domains amino terminal to the core basic region are required for VBP to bind to the full spectrum of PAR target sites. Thus, whereas direct base contacts may be restricted to basic-region residues (as indicated by GCN4-DNA crystal structures), several other domains also influence the DNA-binding specificity of PAR bZIP proteins.

Activating Transcription Factor 2↗

PubMed1995

Cytokine regulation of the liver transcription factor hepatocyte nuclear factor-3 beta is mediated by the C/EBP family and interferon regulatory factor 1.

Three distinct hepatocyte nuclear factor-3 (HNF-3) proteins (alpha, beta, and gamma) regulate the transcription of numerous liver-enriched genes. The HNF-3 proteins bind DNA via a homologous winged helix motif common to a number of proteins known to be critical for determination events in embryogenesis. We have demonstrated previously that two binding sites in the -184 HNF-3 beta promoter are recognized by widely distributed factors and that there is also a critical autoregulatory site, we identified a binding site for a cell-specific factor, LF-H3 beta, that may function in restricting HNF-3 beta gene expression to hepatocytes. Our present study demonstrates that members of the C/EBP and proline and acidic amino acid-rich subfamilies of basic region leucine zipper transcription factors bind the LF-H3 beta site, and cotransfection of HepG2 cells shows that these factors are able to activate an HNF-3 beta promoter reporter construct. The LF-H3 beta-C/EBP binding sequence also confers HNF-3 beta promoter stimulation in response to interleukin (IL)-1 and IL-6. Upstream of this HNF-3 beta proximal promoter region, an IFN-stimulated response element core sequence (-231 to -210) was found that mediates transcriptional induction by IFN-gamma but not IFN-alpha. Gel mobility supershift assay demonstrates that an IFN-gamma-induced protein-DNA complex is disrupted by an antibody specific for interferon regulatory factor-1/interferon-stimulated gene factor-2. Consistent with this finding, we observed that IFN-gamma induction requires ongoing protein synthesis. Surprisingly, the effect of the three cytokines (IL-1, IL-6, and IFN-gamma) in combination as assayed by the same model is not synergistic. HNF-3beta joins the C/EBP family on the list of liver-enriched transcription factors, the expression of which is modulated by cytokines.

Base Sequence↗

PubMed1994

Alternative promoter usage and splicing options result in the differential expression of mRNAs encoding four isoforms of chicken VBP, a member of the PAR subfamily of bZIP transcription factors.

We previously isolated a set of overlapping cDNA clones that encoded a unique open reading frame for the chicken VBP transcription factor. We now report the isolation of a cDNA clone that encodes a complete open reading frame for a VBP isoform that differs from the previously reported sequence at both the amino-terminal and carboxyl-terminal ends. An analysis of the VBP gene revealed that the two different amino-terminal sequences map to alternative first exons and that the two different carboxyl-terminal sequences reflect an optional splicing event which can occur only on transcripts that are polyadenylated at the more distal of two polyadenylation sites. An RT-PCR analysis further revealed that a total of four VBP isoforms are encoded by the combinatorial use of these two splicing options. The mRNAs for these four isoforms are differentially expressed in different tissues and cell types. We provide evidence that one function of the amino-terminal domains is to impose cell type specificity on a core transactivation domain that is present in all four isoforms. Since it is known that VBP can heterodimerize with other members of the PAR subfamily of bZIP factors, our evidence for four VBP isoforms greatly expands the number of complexes that may be used to effect transcriptional regulation through PAR-factor binding sites.

Alternative Splicing↗

PubMed1994

GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut.

We describe experiments which show that: 1) two previously identified members of the GATA family of transcription factors (both of which were originally called GATA-4) correspond to chicken cDNAs for two distinct factors (which we now refer to as cGATA-4 and cGATA-5); 2) another new member of this family corresponds to a third factor designated cGATA-6; 3) each of these mRNAs displays a differential expression pattern. The cGATA-5 gene is initially transcribed in the cardiac crescent prior to formation of the primordial heart tube. Following formation of the primitive heart, cGATA-5 transcripts are evident in both endocardium and myocardium as well as in other lateral plate derivatives. The cGATA-5 gene is also transcribed in the primitive embryonic gut and in late stage embryos is sequentially up-regulated in distinct segments of gastrointestinal epithelia as they undergo terminal differentiation. These studies thus provide novel insights into tissue-specific regulation by GATA-5, as well as into possibly overlapping regulatory functions for these three family members.

Animals↗

PubMed1994

Identification of factor-binding sites in the duck hepatitis B virus enhancer and in vivo effects of enhancer mutations.

Hepatitis B viruses (hepadnaviruses) can cause chronic, productive infections of hepatocytes. Analyses of the enhancers and promoters of these viruses in cell lines have suggested a requirement of these elements for liver-enriched transcription factors. In this study, a minimum of seven factor-binding sites on the duck hepatitis B virus enhancer were detected by DNase I footprinting using duck liver nuclear extracts. Among the sites that were tentatively identified were one C/EBP-, one HNF1-, and two HNF3-binding sites. Mutations of the HNF1- and HNF3-like sites, which eliminated factor binding, as assessed by both DNase I footprinting and competitive gel shift assays, were evaluated for their effects on enhancer activity. Using a construct in which human growth hormone was expressed from the viral enhancer and core gene promoter, we found that all of the mutations, either alone or in combination, reduced expression two- to fourfold in LMH chicken hepatoma cells. The mutations in the HNF1 site and one of the HNF3 sites, when inserted into the intact viral genome, also suppressed virus RNA synthesis in primary hepatocyte cultures. Virus carrying the latter HNF3 mutation was also examined for its ability to infect and replicate in ducks. No significant inhibition of virus replication was observed in a short-term assay; however, virus with the HNF3 mutation was apparently unable to grow in the pancreas, a second site of duck hepatitis B virus replication in the duck.

Animals↗

PubMed1993

Chicken repeat 1 elements contain a pol-like open reading frame and belong to the non-long terminal repeat class of retrotransposons.

Chicken genomes contain approximately 30,000 chicken repeat 1 (CR1) elements scattered among single-copy sequences, but no information has yet been presented to account for how these elements could have dispersed. The fact that CR1 elements have common (although atypical) 3' ends and variable 5' truncations suggested to us that they might belong to the class of non-long terminal repeat retrotransposons that encode reverse transcriptases. From an analysis of unusually large CR1 elements, we now provide evidence for the presence of such a reverse transcriptase open reading frame. CR1 elements are distantly related to previously described non-long terminal repeat retrotransposons; however, we find that frog and torpedo ray genomes contain dispersed open reading frame segments that have > 50% identity to the CR1 open reading frame. This result suggests that CR1-like elements exist in several vertebrate classes that have evolved independently for approximately 400 million years.

Amino Acid Sequence↗

PubMed1992

Isolation of a chicken HMG2 cDNA clone and evidence for an HMG2-specific 3'-untranslated region.

HMG1 and HMG2 (high-mobility group proteins) are two of the most abundant nonhistone chromosomal proteins in higher eukaryotes. Mammalian HMG1 cDNA sequences have the unusual feature of being conserved not only over their coding regions, but also over large segments of their 3'-untranslated regions (3' UTRs) as well. In contrast, the only reported mammalian HMG2 cDNA clone has a distinct 3' UTR. We now report the isolation of a chicken HMG2 cDNA clone and show that it is markedly similar to the mammalian HMG2 cDNA clone over both its coding regions and 3' UTRs. We therefore infer that the 3' UTRs of the HMG1 and HMG2 genes are subject to distinct evolutionary pressures. Our data, along with published data, also serve to highlight 26 amino acid positions where HMG1 and HMG2 are distinctly conserved, and we note that trout HMG-T conforms to the HMG1 paradigm at most of these diagnostic positions.

Amino Acid Sequence↗

PubMed1991

Characterization of the core promoter and enhancer of duck hepatitis B virus.

The core gene promoter of duck hepatitis B virus (DHBV) has been localized and an enhancer element has been found in a region of the DHBV genome immediately upstream of the core promoter. This enhancer was able to activate expression from both the core promoter and the S promoter of DHBV, as well as from the heterologous thymidine kinase and simian virus 40 early promoters, but not from the DHBV PreS promoter, which was active both in the absence and in the presence of the enhancer. The activity of the enhancer showed a preference for cell cultures of hepatic origin, suggesting a possible tissue preference in vivo.

Animals↗

PubMed1991

Chicken vitellogenin gene-binding protein, a leucine zipper transcription factor that binds to an important control element in the chicken vitellogenin II promoter, is related to rat DBP.

We screened a chicken liver cDNA expression library with a probe spanning the distal region of the chicken vitellogenin II (VTGII) gene promoter and isolated clones for a transcription factor that we have named VBP (for vitellogenin gene-binding protein). VBP binds to one of the most important positive elements in the VTGII promoter and appears to play a pivotal role in the estrogen-dependent regulation of this gene. The protein sequence of VBP was deduced from a nearly full length cDNA copy and was found to contain a basic/zipper (bZIP) motif. As expected for a bZIP factor, VBP binds to its target DNA site as a dimer. Moreover, VBP is a stable dimer free in solution. A data base search revealed that VBP is related to rat DBP. However, despite the fact that the basic/hinge regions of VBP and DBP differ at only three amino acid positions, the DBP binding site in the rat albumin promoter is a relatively poor binding site for VBP. Thus, the optimal binding sites for VBP and DBP may be distinct. Similarities between the VBP and DBP leucine zippers are largely confined to only four of the seven helical spokes. Nevertheless, these leucine zippers are functionally compatible and appear to define a novel subfamily. In contrast to the bZIP regions, other portions of VBP and DBP are markedly different, as are the expression profiles for these two genes. In particular, expression of the VBP gene commences early in liver ontogeny and is not subject to circadian control.

Amino Acid Sequence↗

PubMed1991

Mutational studies reveal a complex set of positive and negative control elements within the chicken vitellogenin II promoter.

The endogenous chicken vitellogenin II (VTGII) gene is transcribed exclusively in hepatocytes in response to estrogen. We previously identified two estrogen response elements (EREs) upstream of this gene. We now present an analysis of the VTGII promoter activated by these EREs in response to estrogen. Chimeric VTGII-CAT genes were cotransfected into LMH chicken hepatoma cells along with an estrogen receptor expression vector, and transient CAT expression was assayed after culturing the cells in the absence or presence of estrogen. An analysis of constructs bearing deletions downstream of the more proximal ERE indicated that promoter elements relevant to transcription in LMH cells extend to between -113 and -96. The relative importance of sequences within the VTGII promoter was examined by using 10 contiguous linker scanner mutations spanning the region from -117 to -24. Although most of these mutations compromised VTGII promoter function, one dramatically increased expression in LMH cells and also rendered the VTGII promoter capable of being activated by cis-linked EREs in fibroblasts cotransfected with an estrogen receptor expression vector. Gel retardation and DNase I footprinting assays revealed four factor-binding sites within this promoter. We demonstrate that three of these sites bind C/EBP, SP1, and USF (or related factors), respectively; the fourth site binds a factor that we denote TF-V beta. The biological relevance of these findings is suggested by the fact that three of these binding sites map to sites previously shown to be occupied in vivo in response to estrogen.

Animals↗

PubMed1990

Chromatin studies reveal that an ERE is located far upstream of a vitellogenin gene and that a distal tissue-specific hypersensitive site is conserved for two coordinately regulated vitellogenin genes.

Estrogen induces the expression of three vitellogenin genes in chicken hepatocytes. To survey the vitellogenin III (VTGIII) gene region for possible distal regulatory sequences, we identified tissue-specific hypersensitive (HS) sites within a 45 kb chromatin region spanning this gene. Five constitutive HS sites were found to mark the VTGIII gene region in hormone-naive hepatocytes. Strikingly, the constitutive HS site located 5.5 kb upstream of the VTGIII gene and a previously identified HS site located within the coordinately regulated VTGII gene mapped to nearly identical copies of a 72 bp sequence. Moreover, it would appear that there has been evolutionary pressure to retain specifically this 72 bp of VTGII-like sequence near the VTGIII gene subsequent to the VTGIII and VTGII genes becoming unlinked approximately 16 Myr ago. Two additional sets of HS sites were induced in the VTGIII gene region in response to estrogen. One set mapped immediately upstream of the gene in the vicinity of what we show to be a functional estrogen response element (ERE). The other induced HS site mapped 7.5 kb upstream of the gene. This far-upstream region was sequenced and was found to contain two imperfect ERE consensus sequences spaced 88 bp apart. In transient expression assays neither of these individual imperfect ERE sequences was functional, but a fragment spanning both sequences behaved as a strong ERE. In contrast to this synergism between imperfect ERE sequences, the presence of an NF-1 binding site 23 bp away from the more distal imperfect ERE sequence was not sufficient to render the latter a functional ERE in our assays.

Animals↗

PubMed1990

Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line.

A recently described chicken liver cell line, LMH, was characterized to evaluate responsiveness to estrogen. Expression of the endogenous apolipoprotein (apo) II gene was induced by 17 beta-estradiol when LMH cells were cultured with chicken serum. The response was low and yielded apoll mRNA at only 0.3% of the level seen in estrogenized rooster liver. Higher levels of apoll mRNA were achieved when LMH cells were transiently transfected with an expression plasmid for estrogen receptor. A transfected apoll gene was strongly expressed only when cotransfected with receptor. Expression of the endogenous vitellogenin (VTG) II gene was not detected. However, when cotransfected with a receptor expression plasmid, VTG II reporter plasmids were expressed in LMH cells in response to 17 beta-estradiol. These results suggest that estrogen responsiveness of LMH cells is limited by the availability of functional receptor. Low levels of estrogen receptor mRNA were detected in LMH cells, and receptor binding sites and mRNA were greatly increased following transient transfection with a receptor expression plasmid. Using this transient transfection protocol, several VTG II reporter plasmids were compared in LMH cells and chick embryo fibroblasts. A plasmid containing VTG II estrogen response elements linked to a heterologous promoter was regulated by estrogen in both cell types. In contrast, reporter plasmids containing the VTG II promoter were regulated by estrogen in LMH cells but were not expressed at all in chick embryo fibroblasts. These results suggest that regulation of the VTG II gene involves cell type-specific elements in addition to estrogen response elements.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

The major and minor chicken vitellogenin genes are each adjacent to partially deleted pseudogene copies of the other.

The major chicken vitellogenin gene (VTGII) has previously been cloned and sequenced. We now report the isolation of genomic clones that encompass a minor chicken vitellogenin gene (VTGIII) which is also expressed in the liver in response to estradiol. Our analysis reveals that a pseudogene for VTGII (psi VTGII) lies 1,426 base pairs upstream of this VTGIII gene. A reevaluation of published sequence data reveals that the converse is also true, namely, that a pseudogene for VTGIII (psi VTGIII) lies 1,345 base pairs downstream of the VTGII gene. Our results show that a 335-base-pair deletion has removed the psi VTGIII promoter and cap site but left residual estrogen response element in a region where nuclease-hypersensitive sites have been reported to be induced in response to estradiol.

Animals↗

PubMed1989

Evidence that chicken CR1 elements represent a novel family of retroposons.

We report the first precise delineation of a chicken CR1 element and show that it is flanked by a 6-base-pair target site duplication that occurred when this repetitive element transposed. The 3' end of this CR1 element is defined by an 8-base-pair imperfect direct repeat, and we infer that this sequence represents the 3' end of all intact CR1 elements. In contrast, the 5' ends are not unique, and we argue that this variation existed at the time each element transposed. We also provide evidence that CR1 elements transposed into preferred target sites. CR1 elements therefore appear to represent a novel class of passive retroposons.

Animals↗

PubMed1988

Chronic ethanol or nicotine treatment results in partial cross-tolerance between these agents.

Female DBA/2Ibg mice were treated chronically (21 days) with ethanol- or dextrin-containing liquid diets or infused chronically with nicotine (8 mg/kg/h) or saline for 10 days. The responses of these animals to challenge doses of ethanol (2.5 g/kg) or nicotine (1 or 2 mg/kg) were measured using a test battery consisting of respiration rate, acoustic startle response, Y-maze crosses and rears, heart rate and body temperature. Chronic ethanol-treated animals were tolerant to the effects elicited by a challenge dose of ethanol on four of the six measures and were cross-tolerant to nicotine's effects on the acoustic startle test. Chronic nicotine-treated animals were tolerant to nicotine's effects on five of the six measures and cross-tolerant to ethanol's effects on heart rate and body temperature. Thus, partial cross-tolerance between ethanol and nicotine exists. Chronic nicotine treatment resulted in significant increases in L-[3H]-nicotine binding in six of seven brain regions and in alpha-[125I]-bungarotoxin binding in three of seven brain regions. Chronic ethanol treatment failed to alter the binding of either ligand. Therefore, the cross-tolerance that develops between ethanol and nicotine is not totally dependent on alterations in the number of brain nicotinic receptors.

Animals↗

PubMed1988

Tolerance to and cross tolerance between ethanol and nicotine.

Female DBA mice were subjected to one of four treatments: ethanol-containing or control diets, nicotine (0.2, 1.0, 5.0 mg/kg/hr) infusion or saline infusion. After removal from the liquid diets or cessation of infusion, the animals were challenged with an acute dose of ethanol or nicotine. Chronic ethanol-fed mice were tolerant to the effects of ethanol on body temperature and open field activity and were cross tolerant to the effects of nicotine on body temperature and heart rate. Nicotine infused animals were tolerant to the effects of nicotine on body temperature and rotarod performance and were cross tolerant to the effects of ethanol on body temperature. Ethanol-induced sleep time was decreased in chronic ethanol- but not chronic nicotine-treated mice. Chronic drug treatment did not alter the elimination rate of either drug. Chronic ethanol treatment did not alter the number or affinity of brain nicotinic receptors whereas chronic nicotine treatment elicited an increase in the number of [3H]-nicotine binding sites. Tolerance and cross tolerance between ethanol and nicotine is discussed in terms of potential effects on desensitization of brain nicotinic receptors.

Alcoholism↗

PubMed1988

Isolation of chicken vitellogenin I and III cDNAs and the developmental regulation of five estrogen-responsive genes in the embryonic liver.

The isolation of cDNA clones that code for portions of the two minor chicken vitellogenin (VTG) genes (VTGI and VTGIII) is reported. These clones represent unique sequences that are expressed exclusively in the livers of estrogenized birds. In the liver of the egg-laying hen, the levels of RNAs encoding VTGI, VTGII, and VTGIII are approximately 11,000, 30,000, and 3,000 molecules per cell, respectively. We have used the newly isolated clones, as well as the yolk protein cDNAs previously available [VTGII, apolipoprotein II (apoVLDLII), and apolipoprotein B], as probes to examine several aspects of the regulation of these genes by estradiol. First, we demonstrate that the capacity of each gene to respond to estradiol is acquired between 8 and 13 days in ovo. The response of four of these genes to estradiol is diminished during late fetal development, but the responsiveness is recovered within a week after hatching. Second, we demonstrate that these genes display distinct kinetic response profiles following the addition of estradiol. Third, as has been described previously for the VTGII and apoVLDLII genes, we demonstrate that a single injection of estradiol effects a long-term reprogramming event (hepatic memory) that allows a faster onset of the rapid accumulation of both VTGI and VTGIII RNAs following a subsequent rechallenge by estradiol. Collectively, these three sets of data suggest molecular parameters that may contribute to both the coordinate and noncoordinate regulation of this set of genes by estradiol.

Animals↗

PubMed1988

Two functional estrogen response elements are located upstream of the major chicken vitellogenin gene.

We used a transient-expression assay to identify two estrogen response elements (EREs) associated with the major chicken vitellogenin gene (VTGII). Each element was characterized by its ability to confer estrogen responsiveness when cloned in either orientation next to a chimeric reporter gene consisting of the herpes simplex virus thymidine kinase promoter and the chloramphenicol acetyl transferase-coding region. Deletion analyses indicated that sequences necessary for the distal ERE resided within the region from -626 to -613 (nucleotide positions relative to the VTGII start site) whereas those necessary for the proximal ERE were within the region from -358 to -335. These distal and proximal elements contain, respectively, a perfect copy and an imperfect copy of the 13-base-pair sequence that is an essential feature of the EREs associated with two frog vitellogenin genes. These chicken VTGII EREs mapped near regions that were restructured at the chromatin level when the endogenous VTGII gene was expressed in the liver in response to estradiol. These data suggest a model for the tissue-specific expression of this estrogen-responsive gene.

Acetyltransferases↗